This goal of the proposed research is to combine molecular genetic, cellular and biochemical approaches to gain a fuller understanding of the roles telomere biology plays in human health. Aim #1 investigates in depth specific hypotheses regarding the functional dimerization of telomerase. We will determine the roles of specific residues and domains of the telomerase RNA (TLC1) and TERT (Est2p) in functions of yeast telomerase, including its dimerization and a newly-charactedzed functional protection of telomeres. We found a dynamic pseudoknot structure in telomerase RNAs that is vital to telomerase and its dimerization, and will test a model for dynamic pseudoknot behavior in human telomerase RNA by structural investigations. Aim #2 exploits our new-found ability to highly purify a discrete TAP-tagged Est2p- containing telomerase RNP complex ("telomerase holoenzyme"). The total polypeptide composition (a "telomerase holoenzyme proteome") of the telomerase holoenzyme will be determined. The telomerase holoenzyme was found to contain Tel1p, the yeast ortholog of human DNA damage signaling kinase ATM required for telomere maintenance, and other interesting components: Rad24p, Cdc23p and Mlp1p. We will investigate the composition of telomerase holoenzyme complex(es) through the cell cycle, the dependence of Tel1p association on telomerase core RNP domains, and whether the associations of Tel1p, Rad24p, Cdc23p or Mlp1p with the telomerase complex change in response to telomere length. Electron microscopic analysis of the purified telomerase holoenzyme will be initiated. Aim #3 analyzes the genetic requirements and function of the telomerase deletion response (TDR), a genome-wide expression response specifically elicited by telomerase deletion, and investigates the observation that cells growing in the absence of telomerase are under continual stress. Experiments will investigate the roles of two Est2p-associating proteins that control telomerase action in different ways: Tel1p and PinX1. Protein partners of Tel1p will be identified, and the roles of specific Tel1p domains in augmenting the protection of telomeres by telomerase will be analyzed. Finally, we will explore the new finding that Est2p protein levels are negatively control by the nucleolar protein PinX1. Aim #4 focuses on defining the responses to, and structural differences between, uncapped and capped telomeres. Telomeres will be inducibly modified in defined ways and the short-term cellular and checkpoint responses to these changes will be analyzed, by quantitative readouts. Mechanisms of the responses to specific telomere changes will be explored. Telomere length, composition, or telomedc DNA tract trajectory/conformation of a test telomere will be systematically altered and analyzed in vivo. Based on models to be tested, and using purified telomere components, telomeric complexes will be examined by electron microscopy. [unreadable] [unreadable]